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WHO Protocol for Performance Evaluation of Lymphocyte subsets Enumeration Technologies WHO PQDX_030 v5 June 2017

WHO/EMP/RHT/PQT/2017.xx of 21

WHO PROTOCOL FOR PERFORMANCE

LABORATORY EVALUATION OF

LYMPHOCYTE SUBSETS ENUMERATION

TECHNOLOGIES

WHO Protocol for Performance Evaluation of Lymphocyte subsets Enumeration Technologies WHO PQDX_030 v5 February 2017


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1. Introduction.

1.1 Prequalification of in vitro diagnostics

The World Health Organization (WHO) Prequalification of In Vitro Diagnostics is coordinated through the

Prequalification-Diagnostics Assessment team (PQDx). The aim of the WHO Prequalification of In Vitro

Diagnostics Assessment is to promote and facilitate access to safe, appropriate and affordable diagnostics

of good quality in an equitable manner. Focus is placed on products for high burden diseases and their

suitability for use in resource-limited settings.

The WHO prequalification of in vitro diagnostics process includes three main components:

Review of an pre-submission form and product dossier;

Performance evaluation of the product;

Inspection of the manufacturing site(s).

Performance evaluation will be conducted by two different mechanisms as described at:

http://www.who.int/diagnostics_laboratory/evaluations/alternative/en/. Performance evaluation in List 1

laboratories will be coordinated and paid for by WHO. Performance evaluation in List 2 laboratories will be

coordinated and paid for by the manufacturer.

This protocol provides the details of the procedure for evaluation of Lymphocyte subsets enumeration

technologies submitted for laboratory evaluation as part of the WHO Prequalification of In-vitro Diagnostic

assays on behalf of World Health Organization at the WHO Prequalification Evaluating Laboratory.

This protocol shall not replace more extensive IVD manufacturer’s protocols required for validation and

verification studies of their products.

1.2 WHO Performance evaluation of Lymphocyte subsets enumeration technologies

The performance evaluation determines the accuracy of Lymphocyte subsets enumeration technologies

in comparison with established performance criteria. These characteristics include: repeatability,

reproducibility, accuracy of measurement. In addition, a number of operational characteristics are

assessed including the suitability for use in laboratories and/or testing settings with limited infrastructure.

Dedicated and point-of-care CD4+ T-lymphocytes enumeration technologies used for monitoring of HIV

disease progression in resource limited setting are covered in this protocol

5. Study objectives

Overall objective 5.1.

To conduct multicentre and independent evaluation of dedicated and point-of-care CD4+ T-

lymphocytes enumeration technologies used for monitoring of HIV disease progression in resource

limited settings and disseminate their performance and operation characteristics.

Specific objectives: 5.2.

The specific objectives of the performance evaluation are

http://www.who.int/diagnostics_laboratory/evaluations/alternative/en/



1. To assess repeatability and reproducibility of dedicated and point-of-care CD4+ T-cell enumeration

technologies.

2. To assess the accuracy of measurement of dedicated and point-of-care CD4+ T-cell enumeration

technologies compared to the classical flow cytometer.

3. To determine operational characteristics of dedicated and point-of-care CD4+ T-cell enumeration

technologies.

6. Study design

WHO Prequalification Evaluating Laboratory 6.1.

The WHO Prequalification Evaluating Laboratory shall be one that has undergone assessment using the

WHO Alternative Laboratory Evaluation Mechanism which include submission of an EoI, Stage 1 audit

(Assessment of EoI and specific quality management system (QMS) documentation), Stage 2 audit

which include On-site audit of the laboratory to assess compliance with WHO requirements and lastly

listed as WHO Prequalification Evaluating Laboratories.

http://www.who.int/diagnostics_laboratory/evaluations/alternative/en/.

The laboratory shall hold the following certification for quality management within the laboratory:

ISO17025 (General requirements for the competence of testing and calibration laboratories), ISO15189

(Medical laboratories: Particular requirements for quality and competence) or equivalent.

The person(s) listed in the EoI letter to WHO will act as the Principal Investigator (PI) for the work

performed by the WHO Prequalification Evaluating Laboratory

The laboratory shall hold the following certification for quality management within the laboratory:

ISO17025 (General requirements for the competence of testing and calibration laboratories),

ISO15189 (Medical laboratories: Particular requirements for quality and competence) or equivalent.

The person(s) listed in the EoI letter to WHO will act as the Principal Investigator (PI) for the work performed by the WHO Prequalification Evaluating Laboratory.

http://www.who.int/diagnostics_laboratory/evaluations/alternative/en/



Training performance evaluation and supervision 6.2.

The following issues are key to minimizing error and maximizing the value of this performance

evaluation:

• The PI will be responsible for training the laboratory technicians on the details of the evaluation

protocol and on the performance of each assay undergoing evaluation;

• Only those personnel who have received specific training for this evaluation will be employed in

the evaluation;

• Accurate record keeping is crucial to the success of the evaluation and the PI will be responsible

for ensuring that all data required for the evaluation are recorded on the agreed data collection

sheets, and are accurate and up to date;

• It is important to plan work in advance and follow standard operating procedures as prepared and

controlled by the WHO Prequalification Evaluating Laboratory;

• To reduce the risk of adding an incorrect specimen to a test device/well, before starting the test

run, the operator will prepare worksheets and label all tubes, dilution vessels, test devices or

plates with the specimen’s unique number;

• Because objective, machine-generated, permanent results for simple/rapid diagnostic tests are

not feasible, it is essential that the PI emphasizes to the operator performing the tests the need

for accurate recording of results and recordkeeping;

• To allow immediate correction of erroneous recording of results (rather than differences in visual

interpretation), the PI or designee should assess the results as soon as possible to allow her/him

to return to the original test device to investigate apparently discordant readings;

Safety 6.3.

HIV, hepatitis B and hepatitis C and other viruses are transmissible by blood and body fluids.

Therefore, all types of specimens (including venous and capillary whole blood, serum/plasma, oral

fluid, etc.) must be handled as potentially infectious. Appropriate precautions to minimize infectious

hazards must be taken at all stages from the collection of specimens to the disposal of used materials

from the laboratory. The WHO Guidelines on HIV Safety Precautions, and Guidelines for the Safe

Transport of Specimens (WHO/EMC/97.3) and the WHO Prequalification Evaluating Laboratory

guidelines on laboratory safety should be followed carefully by the laboratory staff.

Storage of assays 6.4.

All reagents must be stored as indicated in the instructions for use. Some assays may not need

refrigeration. If refrigerated storage space is inadequate to store the entire test kit, they may be

divided so that labile reagents can be refrigerated separately from the non-labile supplies. Calibrated

thermometers are placed at each location where reagents and specimens are stored, i.e. ambient,

refrigerator and freezer. Temperatures are recorded daily on the WHO Prequalification Evaluating

Laboratory temperature logs. The lot numbers of the test kits received/used and their expiry dates are

recorded on the individual run worksheets



7. Study samples.

Residual fresh unlinked whole blood samples collected in Ethylene Diamine Tetra acetic Acid (EDTA)

vacutainer tubes submitted for routine CD4 enumeration will be used for the study. However, in those

laboratories where point-of-care CD4 technologies will be evaluated both venous and capillary blood

samples will be used. Efforts will done to include samples from both adults and children with CD4 + T cell

counts ranging from 50 to more than 1000 CD4 T cells/µl of blood in order to cover the clinical decision

range. Samples with higher CD4 values from children less than 6 years will be necessary to determine the

upper analytical limit of the various CD4 technologies being evaluated. Also efforts will be done to include

equal number of males and females, samples from patients with pulmonary tuberculosis, malaria and other

conditions which have been reported to influence CD4 counts. The clinical information including disease

conditions, age, sex and provisional diagnosis will be reported on the data collection form (Appendix 13.1).

Laboratories will be expected to strictly adhere to the 1997 revised Centres for Disease Control and

Prevention (CDC) guidelines for the performing CD4 T cell determinations. The samples will be transported

and maintained at room temperature (18-22oC) before testing unless otherwise specified by the

manufacturer.

8. Sample testing

The WHO Prequalification Evaluating Laboratory will participate in the laboratory testing. The selected

laboratories should have in-house, one of the single platform flow cytometry reference method which for

this study can either be FACSCalibur (BD) or Coulter EPIXS (Beckman Coulter); adherence to Good Clinical

and Laboratory Practice and quality management. The laboratories should have good track record in doing

flow cytometry for CD4+ T lymphocyte determinations and participation in an established external quality

assessment program. During the evaluation all laboratories will be provided with a single batch on CD4

controls which will be run daily and results reported.

The blood samples will be tested in all the technologies following agreed protocol in all the sites and

following the manufacturer's recommendations. In general, all samples will be tested within six to eight

hours of collection by all methods. The reference methods must be well calibrated and all quality control

results should be recorded. The antibody panels for the reference method must contain monoclonal

antibody combinations to accurately enumerate absolute and percent CD3, CD3+CD4+ and CD3+CD8+ T

lymphocytes. One such example is to include a panel with CD3/CD4/CD8/ CD45 monoclonal antibodies. The

stained samples should be analysed immediately after staining, otherwise, they must be stored at 4oC in

the dark and analysed within 24 hours of staining. Two copies of results will be made available from each

sample tested. The extra or scanned copy of the laboratory results will be attached to the clinical form and

sent to WHO Geneva at the end of the study. In those instruments which utilize printer paper which fade

on storage, photocopies of initialized and dated result slips will be made. A maximum of two randomly

selected dedicated CD4 technologies and one reference methods will be evaluated in one laboratory. All

dedicated assays will undergo intra-laboratory variation and agreement between methods in the reference

laboratory. At the end of the study, laboratories will store both the electronic and hard copies of the results

for a minimum of five years or according of local regulations.

Precision measurements 8.1.

Whenever applicable, precision measurements will include repeatability (intra assay variation, and run-to

run variability. Reproducibility will include inter assay variation (day-to-day variability) and inter-instrument

variation. In addition carry-over-studies will be done.

Intra assay variation



To assess intra assay variation will be done on twelve fresh venous whole blood samples. The samples will

be in three groups with 4 samples each in the range 150-250, 300-400 and 450-550 CD4+ T counts/µL. The

samples will be prepared ten times by only one operator, using the same test method and then analysed on

same cytometer. The mean, standard deviation (SD) and percentage coefficient of variation (% CV) will be

calculated and compared.

Run-to-run variation

Run-to-run variation will be assessed in both capillary and venous blood samples by preparing 15 fresh

blood specimen. The samples will be in three groups with 5 samples each in the range 150-250, 300-400

and 450-550 CD4+ T counts/µL. The prepared cartridge will be analysed ten times in a particular instrument

by one operator. The mean, SD and % CV of the obtained results will be calculated and compared

Inter assay variation (day to-day variation)

Inter assay variation will be done on twelve fresh venous whole blood samples. The samples will be in three

groups with 4 samples each in the range 150-250, 300-400 and 450-550 CD4+ T counts/µL. An aliquot from

the samples will be prepared by only one operator at three time points since collection to include 6, 24 and

48 hours and then analysed run on the same cytometer. The mean, SD and % CV of the obtained results will

be calculated and compared

Inter-instrument variation

Inter-instrument variability will be assessed by preparing and analysing 6 fresh venous blood and capillary

samples 10 times on each of the 3-5 analysers. The samples will be in three groups with two samples in the

range 150-250, two samples in the range 300-400 and two samples in the range 450-550 CD4+ T counts/µL.

For each sample, 10 cartridges will be prepared, and each cartridge will be read on each of the study

instruments. The five samples can be analysed by different operators in order to evaluated inter-operator

variability. The mean, SD and % CV of the obtained results will be calculated and compared.

The acceptability criteria for the four precision studies is shown in table 1 below

Carry-Over-studies

This will be measured by analysing five batches of two identical blood specimens with a high CD4 counts

greater than 600/µL (recorded as al and a2) followed by two identical blood specimens with CD4 counts

between 100 and 300/µL (which are recorded as b1 and b2).

(A1 A2 B1 B2). The carry-over (k) is expressed as described by Broughton.

%100b - a

b - b

22

21 k

The mean result should be the same for high-low and low-high sequences. With most automatic analysers,

carry-over is less than 2%.

Table 1 The optimal/desirable precision measurements criteria for dedicated and POC CD4

technologies

CD4 levels Coefficient

of variation

Standard

deviation

95% CI

Intra-assay variability ~200/µl <15% <30cells/µl 21, 55



~350/µl <10% <35cells/µl 24, 64

~500/µl <10% <50cells/µl 34, 91

inter-instrument variability ~200/µl <15% <30cells/µl 24, 40

~350/µl <10% <35cells/µl 28, 47

~500c/µl <10% <50cells/µl 40, 67

inter- assay variability ~200/µl <15% <30cells/µl 24, 40

~350/µl <10% <35cells/µl 28, 47

~500c/µl <10% <50cells/µl 40, 67

Trueness of measurement (Agreement between methods) 8.2.

Consecutive routine blood samples in EDTA vacutainer tubes with at least 3.0 ml of blood brought into the

laboratory (and capillary blood collected directly from the patient where applicable) will be used to

compare the methods. In each site a total of 200 samples of which 50 will have CD4+T cell range of <200/µL,

100 samples will have CD4 counts between 201 and 500/µL and the remaining 50 blood samples will

have >500 CD4+ T lymphocytes/µL will be stained by the test method and the reference method and run in

the respective instruments. Agreement between the dedicated and the reference method will be assessed

using the Bland Altman plots and/or percentage similarity methods. The overall bias should be less than 10%

with SD shown in Table 2 below

Table 2. The minimum and optimal performance criteria (bias) for instrument trueness of measurement.

Minimum Optimal

CD4 levels Number of

samples

Standard

Deviation

Width of

95% CI

Number of

samples

Standard

Deviation

Width of

95% CI

<200/µl 50 40cells/µl 23cells/µl 50 30 cells/µl 17cells/µl

200 -500/µl 100 50 cells/µl 20cells/µl 100 35 cells/µl 14cells/µl

>500c/µl 50 75 cells/µl 43cells/µl, 50 50 cells/µl 28cells/µl

Safety 8.3.

The testing of all specimens should be performed following WHO guidelines for good laboratory practice to

minimize occupational exposure risks. [For further details see Laboratory Biosafety Manual 3rd ed., Geneva,

World Health Organization, 2004 (ISBN 9241546506), Biosafety Guidelines for Diagnostic and Research

Laboratories Working with HIV, Geneva, World Health Organization, 1991 (WHO AIDS Series 9) and

Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens, Geneva, World Health

Organization, 1997 (WHO/EMC/97.3) ].



9. Ethical considerations

The protocol will be submitted to WHO Ethical Review Committee, National and Institutional ethical review

boards in each participating country for ethical clearance. Residual routine patient blood samples which

have been submitted to the laboratories for enumeration of CD4+ T lymphocytes as part of routine

management will be used in the study. However in those sites where point-of-care CD4 technologies which

require capillary blood will be evaluated an informed consent will be obtained from the patients (Annex

13.3). Patient's results will be issued as per standard operating procedures (SOP) effective in each of the

study sites. The study will not interfere with the patients results as the study results will be unlinked. Age

and sex of the patient will be recorded in the study form and all other patient's identifiers will then be

removed from the sample tube, and given a new identification number. Results obtained from the CD4

technologies being evaluated will not be used for patient's management. The following clinical information

will also be collected from the patient's forms; patient's provisional clinical diagnosis, such tuberculosis,

malaria or HIV. It is presumed that when the above measures are taken it may not be necessary to request

informed verbal or written consent from the patients during blood collection and ethical review board will

be requested to waive the need for informed consent. The manufacturers of the CD4 technologies will be

provided with the protocol before the commencement of the study

10. Data management and Statistical analysis

Laboratory technologists performing the assay will also complete a standard form detailing its operational

characteristics relevant to resource limited settings (Annex 13.4), daily control log and adverse event log

(Annex 13.5). The individual laboratories will enter the data in the provided excel sheets under the

supervision of the Principal Investigator (Annex 13.6). A scanned copy of all data collection forms will be

also be sent to WHO/DLT Geneva for verification and further analysis. Data from individual laboratories will

be verified and combined into one data set for analysis at WHO HQ, Geneva.

Precision of the technology will be determined by calculation of the mean, standard deviation and

coefficient of variation as defined below.

The mean (x) is a measure of central tendency of a set of values.

The standard deviation (SD) is a measure of spread of a set of measurements about the mean.

The coefficient of variation (CV) expresses the standard deviation as a percentage of the mean.

Agreement between methods which is the estimates of analytical errors can be obtained by calculating

the bias using the modified Bland-Altman analysis and/or the “percentage similarity scatter plot” by

Scott et al.

The test method must demonstrate good precision as indicated in the respective section in the study

design above.

11. Time frame

The evaluations will be expected to be completed within 3 months.

12. Communications

Principal Investigators of the participating laboratories will be in constant communication with the WHO

HQ PQ Diagnostics Assessment team in Geneva to follow-up the progress during the whole process of

evaluation. The preliminary report on the CD4 technologies will be shared with individual manufactures,

who will be required to file any disagreements within one month. The results will be presented in



scientific forum, published in international scientific journal and will also be posted on the WHO/DLT

website. The prequalified products will also be included in the WHO WebBuy.

13. Responsibilities of the partners.

Manufacturers of the CD4 technologies 13.1.

Will provide the instruments and appropriate number of kits (about 500 tests per site) free-of-charge

for the study;

Ensure that they ship the instruments to the testing laboratory under appropriate conditions and in

time for the commencement of the evaluation;

Ensure that all test kits have at least 6 months remaining shelf life at the start of the evaluation.

Storage conditions during transport must be recorded;

Carry out training on the use of the instruments for the laboratory staff carrying out the evaluation;

Ensure prompt instrument service when there is breakdown.

Agree to sign a contract provided by WHO.

Respond to WHO their concerns regarding the evaluation results of their technologies within one

month.

WHO Prequalification Testing Laboratory 13.2.

Sign WHO contract for performance of work

Declare any conflict of interest

Will submit the agreed protocol to the national bodies for ethical clearance ;

Liaison with manufacturer regarding appropriate date(s) for delivery and installation of the instruments

and associated test kits;

Ensure all specimens are properly processed as specified in the protocol including strict quality

assurance for all the methodologies under evaluation;

Ensure that results are stored securely;

Ensure that instruments are evaluated according to the protocol;

Ensure that testing records are complete and accurate;

Fill the patient information form and attach copies of results from the instrument printer have been

initialized and dated;

Perform data entry and ease of use of the technology;

Ensure confidentiality of the result.

Diagnostics and Laboratory Technologies 13.3.

Submit the protocol to WHO ethical clearance committee;

Finalize formal contracts with CD4 companies and the participating laboratories;

Will be the body which is allowed to communicate with the companies once the study start

Provide technical and administrative support of the project;

Ensure availability of funds for procurement and delivery of all essential supplies required for the

reference method;

Supervise preparation and dissemination of the final report;

Perform final analysis and publish the results.



14. References

1. CDC. 1997 Revised guidelines for performing CD+ T- cell determinations in persons infected with Human

Immunodeficiency Virus (HIV) MMWR 1997;6 (RR-2):-29.

2. NCCLS, Evaluation of Precision Performance of Quantitative Measurement Methods; Approved

Guideline— Second Edition EP5-A2

3. Chesher D. Evaluating assay precision. Clinical Biochemistry Review. 2008 Aug;29 Suppl 1:S23-6.

4. Broughton PM, Gowenlock AH, McCormack JJ, Neill DWA. Revised scheme for the evaluation of

automatic instruments for use in clinical chemistry. Ann Clin Biochem. 1974 Nov;11(6):207-18.

5. Martin Bland J., Douglas G. Altman. Statistical methods for assessing agreement between two methods

of clinical measurement. Lancet, 1986; i: 307-310

6. Scott LE, Galpin JS, Glencross DK. Multiple method comparison: statistical model using percentage

similarity. Cytometry B Clinical Cytometry. 2003 Jul;54(1):46-53.

http://www.ncbi.nlm.nih.gov/pubmed/18852851?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Broughton%20PM%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Gowenlock%20AH%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22McCormack%20JJ%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Neill%20DW%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/pubmed/12827667?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

http://www.ncbi.nlm.nih.gov/pubmed/12827667?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum



13. Annexes

13.1. Patient clinical information form

Performance evaluation of dedicated CD+ T lymphocytes enumeration technologies

Name of the evaluation site ………………………………………………………….

Date …………………………

Sample ID …………………………

Time of collection o blood sample …………………………

1. Age of the patient (years) ………………………………………..

2. Sex ………

3. Provisional Diagnosis ………………………………………..

4. Current patient medication ………………………………………..

a. Patient on ARV YES NO UNKNOWN

5. Patient has TB YES NO NO UNKNOWN

6. Patient has Malaria YES NO UNKNOWN

Initials of the technologist Initials of the validating PI

____________________ ____________________

Date Date

________________ __________________

Note: Please attach a copy of the print out from the CD4 instrument

MALE

FEMALE



13.2. Summary of the experimental protocol

*INTRA

LABORAT

ORY

*Sample

type

Sample

size

CD4 T-

cell

strata

Details Remark

Assay

precision

Intra

Assay

Tube-

to-

Tube

Venous 4 150-250 10

cartridges/sa

mple, each

sample on a

single

instrument

1 out of the 4 samples

acceptable on a 75-cell

distance from threshold in

place of 50 (125-275 for 200;

275-425 for 350, and 425-575

for 500)

4 300-400

4 450-550

Total=12

Inter

Assay

Assay-

to-

Assay

Venous 4 150-250 6 hours, 24

hours, 48

hours




place of 50 (125-275 for 200;

275-425 for 350, and 425-575

for 500)

4 300-400

4 450-550

Total=12

Instrumen

t

precision

Run-

to-

Run

Venous

and

Capillary

5 150-250 10 runs of one

cartridge (on

the same

instrument)




place of 50 (125-275 for 200;

275-425 for 350, and 425-575

for 500)

5 300-400

5 450-550

Total=15

Inter-

instru

ment

Venous

and

Capillary

2 150-250 10

cartridges/sa

mple, each

sample on

each of the 4

instruments (3

in study + 4th

in stock)

2 300-400

2 450-550

Total=6

Carry-

over

Carry-

over

Venous 5 100-300 Sequence

High-high-

low-low

* May not be applicable in

most of POC assays

5 >600

Total=10

INTER

LABORAT

ORY

Inter

lab

Venous

and

Capillary

50 <200 Run in the

POC and

reference

method

100 201-500

50 >500

Total 200

*Sample types and type of studies will depend on the type of technology to be evaluated



13.3. Informed Consent Form

[YOUR INSTITUTIONAL LETTERHEAD]

Informed Consent for Collection of Blood in a multi-centre evaluation of dedicated and point of care CD4

technologies

[Name of Principle Investigator] ………………………………………………..

[Name of Principal Investigator] ………………………………………………..

[Name of Organization] ………………………………………………..

[Name of Sponsor] World Health Organization

This Informed Consent Form has two parts:

1. Information Sheet (to share information about the research with you)

2. Certificate of Consent (for signatures if you agree to take part)

You will be given a copy of the full Informed Consent Form

PART I: Information Sheet

Introduction

I am …….., working for ………… [Name of the hospital]. We are doing an evaluation of instruments used in

monitoring patients with human immunodeficiency virus (HIV) infection, which is very common in this

country. I am going to give you information and invite you to participate in this research. You do not have

to decide today whether or not you are willing to participate in the research. Before you decide, you can

talk to anyone you feel comfortable with about the research. There may be some words that you do not

understand. Please ask me to stop as we go through the information and I will take time to explain. If you

have questions later, you can ask me, the study staff.

Purpose of the research

HIV is one of the most common diseases in this region. It is important for health care providers to

determine the number of specific blood cells in order to accurately determine the time to initiate various

treatment options and also monitor response to treatment. Specific instruments are required to count

these blood cells. We are doing this research to evaluate the ability of newly developed instruments to

accurately count the cells.

Type of Research Intervention

This research will involve measuring blood from various patients in two or more instruments and then

compare the results.

Participant selection

We are inviting all patients attending this clinic to participate in the research to evaluate the performance

of the instrument. Your participation in this research is entirely voluntary. It is your choice whether to give

a blood sample or not. Whether you choose to participate or not, all the services you receive at this clinic

will continue and nothing will change. You may change your mind later and stop participating even if you

agreed earlier.

Procedures and Protocol

About three able spoons of blood will be collected from the arm using a syringe. In addition we will take a

drop of blood from your finger tip. We will take your blood only once.



Risks

We do not expect that any harm will happen to you because of joining this study. Sometimes, you may feel

some little pain and a small bruise may occur due to the finger prick.

Benefits

If you participate in this research, you will not have immediate individual benefits but your participation is

likely to help us ensure availability of better and cheaper service to patients in the future.

Reimbursements

You will not be given any money or gifts to take part in this research.

Confidentiality

The information that will be collected from this research project will be kept confidential. Information

about you that will be collected during the research will be put away and no-one but the researchers will be

able to see it. Any information about you will have a number on it instead of your name. Only the

researchers will know what your number is and we will lock that information up with a lock and key. It will

not be shared with or given to anyone except [name] who will have access to the information, such as

research sponsors, etc.)

Right to Refuse

You do not have to take part in this research if you do not wish to do so and refusing to participate will not

affect your treatment at this clinic in any way. You will still have all the benefits that you would otherwise

have at this clinic.

Who to Contact

If you have any questions you may ask them now or later, even after the study has started. If you wish to

ask questions later, you may contact any of the following: [name, address/telephone number/e-mail]

This proposal has been reviewed and approved by [name of the local IRB], which is a committee whose task

it is to make sure that research participants are protected from harm. If you wish to find about more about

the IRB, contact [name, address, telephone number.]. It has also been reviewed by the Ethics Review

Committee of the World Health Organization (WHO), which is funding/sponsoring/supporting the study.

You can ask me any more questions about any part of the research study, if you wish to. Do you have any

questions?

PART II: Certificate of Consent

I have read the foregoing information, or it has been read to me. I have had the opportunity to ask

questions about it and any questions that I have asked have been answered to my satisfaction. I consent

voluntarily to participate as a participant in this research.

Name of Participant__________________

Signature of Participant ___________________

Date ___________________________ (Day/month/year)

If illiterate



I have witnessed the accurate reading of the consent form to the potential participant, and the individual

has had the opportunity to ask questions. I confirm that the individual has given consent freely.

Name of witness_____________________ AND Thumb print of participant

Signature of witness ______________________

Date ________________________ (Day/month/year)

Statement by the researcher/person taking consent

I have accurately read out the information sheet to the potential participant, and to the best of my ability

made sure that the participant understands that the following will be done:

1. We will collect few mls of blood using venepuncture

2. We will also collect capillary blood using the finger tip

I confirm that the participant was given an opportunity to ask questions about the study, and all the

questions asked by the participant have been answered correctly and to the best of my ability. I confirm

that the individual has not been coerced into giving consent, and the consent has been given freely and

voluntarily. A copy of this ICF has been provided to the participant.

Name of Researcher/person taking the consent________________________

Signature of Researcher /person taking the consent__________________________

Date ___________________________ (Day/month/year)

13.4. Assessment of operational characteristics of CD4 Technology

Operational characteristic Options Yes/No

Characteristics of the technology

Technology format

Portable bench top instrument

Hand-held instrument

Instrument free test

Instrument Calibration Auto-calibration or calibration not required

Portability Transportable by back pack (<10Kg)

Transportable by hand (<2 Kg)

In-built software Present (for instrumentation)

Data capture capability (instrument free)

Instrument Connectivity Universal communication protocol (USB)

Wireless Data transfer

Power Source (when needed or

applicable)

Mains and rechargeable battery

Rechargeable battery

Alternate Power Sources Solar energy, car battery

Lockout instrument features Administrative control of user, expired test lots, error

(critical system check failure shutdown, internal QC

failure,)

Waste

Minimal solid waste (biohazard material contained)

Minimal liquid and solid waste



Result Display

Permanent record (displayed on the device and

printed)

Electronic captured result

Result Storage Results stored in the instrument and there is an

interface

External Equipment(s) Not required

Frequency of breakdown/

blockage over the study period (1-

3 months)

None

1-5

>5

Technology output characteristics

Assay output CD4 counts

CD4 counts and percentage

Multiplex capability

Analytical range for quantitative

variables

0-1000 CD4 cell/µl

0-2000 CD4 cells/µl

Sample throughput 5-15 per day per instrument per operator

Turn-around-time ~30 minutes

<10 minutes

Quality control

Internal Quality Control available Yes

Assay run controls available Yes

External Quality Control(EQA Compatible with a recognised EQA samples

System check (instrument) Available

Reagents

Instruction for use Concise and clear

Heat Stability of Reagents and

Controls

Less than 30oC for minimum of 12 months

Max 40oC degrees for minimum of 6 months

Shelf life of reagents upon

manufacture

12 Months at room temperature (18-25oC)

>12 Months

Reagent & Control Preparation Not required

Sample preparation and analysis

Type of Sample Venous blood

Capillary blood

Venous and capillary blood

Sample collection Not require specialised tube

No need of tubes

Sample Labelling options Manual label

Bar code reader

Precise Sample Measurement No manual pipetting of precise quantity required

Specimen volume required Maximum 100µl

<50ul

Stability of the stored blood Can be tested up to 8 hours



specimen Can be tested up to 48 hours

Stability after staining/adding to

cartridge/devise

0 – 8 hours

Sample failure rates Failure rates <5%

Failure rate <3%

Regulatory and operational issues

Service and Maintenance Easily available in-country/region

Rent or lease agreement Available

Capital Cost of Equipment Less than $1,000

Reagent, Consumables, controls

cost

Less than $5 cost per test

Connectivity cost Included in cost per test/equipment

Operator skills required Trained health care worker

Installation Do not require Vendor or Technician

Operating environment Wide operating range for temperature humidity

altitude, dust

Preventative Maintenance All routine maintenance by Operator

No routine maintenance

Training Maximum 1 day if formal training required

<0.5days maximum if formal training required

Supply Chain and Distribution Easily available in-country/region

Test availability and Regulatory

Status

Commercially available and approved in-

country/region

Best suited for Health centre/Dispensary

13.5. Adverse events reporting log

WHO multicentre evaluation of CD4 technologies. Adverse event reporting log

Name of the laboratory____________________________________________

Name of the instrument___________________________________________

Date Details of the event Corrective action

done

Lab technician

initial and date

Supervisor

initials and

date



13.6. Data collection template (Using Excel sheet)

INTRA-ASSAY VARIATION

Sample Aliquots

CD3

counts

CD3

%

CD4

counts

CD4

%

CD8

counts

CD8

%

A1

A2

A3

A4

A5

A6

A7

A8

A9

INTER ASSAY VARIATION COUNTS

SIX HOURS 24 HOURS 48 HOURS

Sample

CD3

counts

CD4

counts

CD8

counts

CD3

counts

CD4

counts

CD8

counts

CD3

counts

CD4

counts

CD8

counts

B1

B2

B3

B4

B5

B8

B9

B10

INTER ASSAY VARIATION PERCENTAGES

SIX HOURS 24 HOURS 48 HOURS

sample CD3% CD4% CD8% CD3% CD4% CD8% CD3% CD4% CD8%

B1

B2

B3

B4

B5

B8

B9

B10



Precision

CD3

counts

CD3

%

CD4

counts

CD4

%

CD8

counts

CD8

%

1st run

2nd run

3rd run

4th run

5th run

6th run

7th run

8th run

Carry over

Sequence

CD3

counts

CD3

%

CD4

counts

CD4

%

CD8

counts

CD8

%

3

3

1

1

3

3

3

2

2

2

1

1

1

AGREEMENT WITH THE REFERENCE METHOD (For each method)

CD3

counts

CD3

%

CD4

counts

CD4

%

CD8

counts

CD8

%

1

2

3

4

5

6

7

11

74



QUALITY CONTROL RESULTS (For each method)

Dates

CD3

counts

CD3

%

CD4

counts

CD4

%

CD8

counts

CD8

%

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